Carbon-substituted diketopiperazine delivery systems

ABSTRACT

Compositions useful in the delivery of active agents are provided. These delivery compositions include (a) an active agent; and (b) a carrier of at least one mono-C-substituted or di-C-Substituted diketopiperazine. Methods for preparing these compositions and administering these compositions are also provided.

FIELD OF THE INVENTION

[0001] The present invention relates to compositions for deliveringactive agents, and particularly biologically active agents. The carriersin these compositions facilitate the delivery of a cargo to a target.These delivery compositions are particularly useful in the oral deliveryof biologically active agents such as pharmacologically ortherapeutically active agents. Methods for the preparation and for theadministration of such compositions are also disclosed.

BACKGROUND OF THE INVENTION

[0002] Conventional means for delivering active agents are oftenseverely limited by biological, chemical, and physical barriers.Typically, these barriers are imposed by the environment through whichdelivery occurs, the environment of the target for delivery, or thetarget itself. Biologically active agents are particularly vulnerable tosuch barriers.

[0003] For example in the delivery to animals of pharmacological andtherapeutic agents, barriers are imposed by the body. Examples ofphysical barriers are the skin and various organ membranes that must betraversed before reaching a target.

[0004] Chemical barriers include, but are not limited to, pH variations,lipid bi-layers, and degrading enzymes.

[0005] These barriers are of particular significance in the design oforal delivery systems. Oral delivery of many biologically active agentswould be the route of choice for administration to animals if not forbiological, chemical, and physical barriers such as varying pH in thegastrointestinal (GI) tract, powerful digestive enzymes, and activeagent impermeable gastrointestinal membranes.

[0006] Among the numerous agents which are not typically amenable tooral administration are biologically active peptides, such as calcitoninand insulin; polysaccharides, and in particular mucopolysaccharidesincluding, but not limited to, heparin; heparinoids; antibiotics; andother organic substances. These agents are rapidly rendered ineffectiveor are destroyed in the gastrointestinal tract by acid hydrolysis,enzymes, or the like.

[0007] Earlier methods for orally administering vulnerablepharmacological agents have relied on the co-administration of adjuvants(e.g., resorcinols and non-ionic surfactants such as polyoxyethyleneoleyl ether and n-hexadecylpolyethylene ether) to increase artificiallythe permeability of the intestinal walls, as well as theco-administration of enzymatic inhibitors (e.g., pancreatic trypsininhibitors, diisopropylfluorophosphate (DFF) and trasylol) to inhibitenzymatic degradation.

[0008] Liposomes have also been described as drug delivery systems forinsulin and heparin. See, for example, U.S. Pat. No. 4,239,754; Patel etal. (1976), FEBS Letters, Vol. 62, pg. 60; and Hashimoto et al. (1979),Endocrinology Japan, Vol. 26, pg. 337.

[0009] However, broad spectrum use of such drug delivery systems isprecluded because: (1) the systems require toxic amounts of adjuvants orinhibitors; (2) suitable low molecular weight cargos, i.e. activeagents, are not available; (3) the systems exhibit poor stability andinadequate shelf life; (4) the systems are difficult to manufacture; (5)the systems fail to protect the active agent (cargo); (6) the systemsadversely alter the active agent; or (7) the systems fail to allow orpromote absorption of the active agent.

[0010] More recently, microspheres of artificial polymers of mixed aminoacids (proteinoids) have been used to deliver pharmaceuticals. Forexample, U.S. Pat. No. 4,925,673 describes drug-containing proteinoidmicrosphere carriers as well as methods for their preparation and use.These proteinoid microspheres are useful for the delivery of a number ofactive agents.

[0011] There is still a need in the art for simple, inexpensive deliverysystems which are easily prepared and which can delivery a broad rangeof active agents.

SUMMARY OF THE INVENTION

[0012] Compositions useful in the delivery of active agents areprovided.

[0013] These delivery compositions comprise (a) an active agent; and (b)a carrier comprising at least one mono-C-substituted or di-C-substituteddiketopiperazine.

[0014] Biologically active agents and pharmacologically active agentsmay be incorporated as the active agent, and these compositions may bein the form of microspheres.

[0015] Also contemplated is a method for preparing these compositionswherein at least one active agent is mixed with a carrier as describedabove or wherein the carrier is solubilized in a solvent, and thecarrier solution is contacted with the active agent and a precipitatorsolution in which the carrier is insoluble.

[0016] In a further embodiment, the compositions are administered,preferably orally, to animals.

BRIEF DESCRIPTION OF THE DRAWING

[0017]FIG. 1 is an illustration of a reaction scheme for the preparationof diketopiperazines.

DETAILED DESCRIPTION OF THE INVENTION

[0018] The present invention is suited to the delivery of any activeagents through various biological, chemical, and physical barriers. Itis particularly suited to the delivery of active agents which aresubject to environmental degradation.

[0019] Other advantages provided by the present invention include theuse of readily available or easy to prepare, inexpensive startingmaterials. The formulation methods of the present invention arecost-effective for preparing and isolating these compositions, aresimple to perform, and are amenable to industrial scale up forcommercial production.

[0020] Active Agents

[0021] Active agents suitable for use in the present invention includebiologically active agents, chemically active agents, including, but notlimited to, fragrances, as well as other active agents such as, forexample, cosmetics.

[0022] Biologically active agents include, but are not limited to,pesticides, pharmacological agents, and therapeutic agents. For example,biologically active agents suitable for use in the present inventioninclude, but are not limited to, peptides, and particularly smallpeptides; hormones, and particularly hormones which by themselves do notor only pass slowly through the gastrointestinal mucosa and/or aresusceptible to chemical cleavage by acids and enzymes in thegastrointestinal tract; polysaccharides, and particularly mixtures ofmuco-polysaccharides; carbohydrates; lipids; or any combination thereof.Further examples include, but are not limited to, human growth hormones;bovine growth hormones; growth releasing hormones; interferons;interleukin-1; insulin; heparin, and particularly low molecular weightheparin; calcitonin; erythropoietin; atrial naturetic factor; antigens;monoclonal antibodies; somatostatin; adrenocorticotropin, gonadotropinreleasing hormone; oxytocin; vasopressin; cromolyn sodium (sodium ordisodium chromoglycate); vancomycin; desferrioxamine (DFO);anti-microbials, including, but not limited to anti-fungal agents; orany combination thereof.

[0023] The compositions of the present invention may include one or moreactive agents.

[0024] Diketopinerazines

[0025] The diketopiperazines of the present invention are six memberring compounds. The ring includes two nitrogen atoms and is substitutedat two carbons with two oxygen atoms. Preferably, the carbonyl groupsare at the 1 and 4 ring positions. These rings are further substitutedat either or both of the other two carbon atoms of the ring.

[0026] Most preferred diketopiperazines are compounds of the formula:

[0027] wherein R and R¹ independently are hydrogen, C₁-C₂₄ alkyl, C₁-C₂₄alkenyl, phenyl, naphthyl, (C₁-C₁₀alkyl)phenyl, (C₁-C₁₀alkenyl)phenyl,(C₁-C₁₀ alkyl)naphthyl, (C₁-C₁₀ alkenyl)naphthyl, phenyl (C₁-C₁₀ alkyl),phenyl(C₁-C₁₀ alkenyl), naphthyl (C₁-C₁₀ alkyl), and naphthyl (C₁-C₁₀alkenyl); but both R and R¹ can not be hydrogen; either or both R or R¹independently may optionally be substituted with C₁-C₄ alkyl, C₁-C₄alkenyl, C₁-C₄ alkoxy, —OH, —SH, and —CO₂R² or any combination thereof;R² is hydrogen, C₁-C₄ alkyl or C₁-C₄ alkenyl; and either or both R andR¹ independently may optionally be interrupted by oxygen, nitrogen,sulfur, or any combination thereof.

[0028] The phenyl or naphthyl groups may optionally be substituted.Suitable, but non-limiting, examples of substituents are C₁-C₆ alkyl,C₁-C₆ alkenyl, C₁-C₆ alkoxy, —OH, —SH, or CO₂R³ wherein R³ is hydrogen,C₁-C₆ alkyl, or C₁-C₆ alkenyl. When one of R or R¹ is hydrogen, thediketopiperazine is mono-carbon-(C)-substituted. When neither R nor R¹is hydrogen, the diketopiperazine is di-carbon-(C)-substituted.Preferably, R, R¹, or both R and R¹, contain at least one functionalgroup, a functional group being a non-hydrocarbon portion responsiblefor characteristic reactions of the molecule. Simple functional groupsare heteroatoms including, but not limited to halogens, oxygen, sulfur,nitrogen, and the like, attached to, the carbon of an alkyl group by asingle or multiple bond. Other functional groups include, but are notlimited to, for example, hydroxyl groups, carboxyl groups, amide groups,amine groups, substituted amine groups, and the like.

[0029] Preferred diketopiperazines are those which are substituted atone or two of the carbons of the ring with a functional group thatincludes at least one carboxyl functionality.

[0030] Diketopiperazines typically are formed from a-amino acids. The“term” amino acid used with respect to diketopiperazines also includesany carboxylic acid having at least one free α-amine group and includesnaturally occurring and synthetic α-amino acids and all optical isomersthereof. Preferably, the diketopiperazines are formed from two aminoacids which are the same or optical isomers of one another. Typicalamino acids useful in the preparation of diketopiperazines are naturalor synthetic amino acids having the formula:

HN(R⁴)—(R⁵)—OH  II

[0031] R⁴ is hydrogen, C₁ to C₂₄ alkyl, C₁-C₂₄ alkenyl, phenyl,naphthyl, (C₁-C₁₀ alkyl) phenyl, (C₁-C₁₀ alkenyl) phenyl, (C₁-C₁₀ alkyl)naphthyl, (C₁-C₁₀ alkenyl) naphthyl, phenyl (C₁-C₁₀ alkyl), phenyl(C₁-C₁₀ alkenyl), naphthyl (C₁-C₁₀ alkyl), and naphthyl (C₁-C₁₀alkenyl); optionally R⁴ may be substituted with C₁-C₄ alkyl, C₁-C₄alkenyl, C₁-C₄ alkoxy, —OH, —SH, —CO₂R⁶, or any combination thereof; R⁶is hydrogen C₁-C₄ alkyl, or C₁-C₄ alkenyl; and R₄ may optionally beinterrupted by oxygen, nitrogen, sulfur, or any combination thereof.

[0032] The phenyl or naphthyl groups may optionally be substituted.Suitable, but non-limiting examples of substituents are C₁-C₆ alkoxy,—OH, —SH, or CO₂R⁷, wherein R⁷ is hydrogen C₁-C₆ alkyl, or C₁-C₆alkenyl.

[0033] R⁵ has the formula

[0034] wherein R⁸ is C₁ to C₂₄ alkyl, C₁ to C₂₄ alkenyl, phenyl,naphthyl, (C₁ to C₁₀ alkyl)-phenyl, (C₁ to C₁₀ alkenyl)phenyl, (C₁ toC₁₀ alkyl)naphthyl, (C₁ to C₁₀ alkenyl)naphthyl, phenyl (C₁ to C₁₀alkyl), phenyl (C₁ to C₁₀ alkenyl), naphthyl (C₁-C₁₀ alkyl), andnaphthyl (C₁-C₁₀ alkenyl); R⁸ may optionally be substituted with C₁ toC₂₄ alkyl or C₁ to C₂₄ alkenyl; and R⁵ may optionally be interrupted byoxygen, nitrogen, sulfur or any combination thereof.

[0035] The phenyl or naphthyl groups may optionally be substituted.Suitable but non-limiting examples of substituents are C₁ to C₆ alkyl,C₁ to C₆ alkenyl, C₁-C₆ alkoxy, hydroxy, thio, or CO₂R⁹ alkenyl, whereinR⁹ is hydrogen, C₁-C₆ alkyl, or C₁-C₆ alkenyl.

[0036] The preferred naturally occurring amino acids for preparation ofthe diketopiperazines of the present invention are alanine, arginine,asparagine, aspartic acid, citrulline, cysteine, cystine, glutamine,glycine, histidine, isoleucine, leucine, lysine, methionine, ornithine,phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine,or hydroxy proline.

[0037] The preferred non-naturally occurring amino acids for use hereinare phenylglycine, α-aminobutyric acid, α-amino isobutyric acid,aminobenzoic acid, aminohippuric acid, and cysteine.

[0038] Preferably, the α-amino acids of which the diketopiperazines arederived are glutamic acid, aspartic acid, tyrosine, phenylalanine, andoptical isomers of any of the foregoing. Most preferably, thediketopiperazines useful in the present invention are prepared fromtrifunctional amino acids such as L-glutamic acid and L-aspartic acidwhich cyclize to form diketopiperazines.

[0039] Dipiperazine ring systems may be generated during thermalpolymerization or condensation of amino acids or amino acid derivatives.(Gyore, J; Ecet M. Proceedings Fourth ICTA (Thermal Analysis), 1974, 2,387-394 (1974)). These six membered ring systems were presumablygenerated by intra-molecular cyclization of the dimer prior to furtherchain growth or directly from a linear peptide (Reddy, A. V., Int. J.Peptide Protein Res., 40, 472-476 (1992); Mazurov, A. A. et al., Int. J.Peptide Protein Res., 42, 14-19 (1993)).

[0040] Diketopiperazines can also be formed by cyclodimerization ofamino acid ester derivatives as described by Katchalski et al., J. Amer.Chem. Soc., 68, 879-880 (1946), by cyclization of dipeptide esterderivatives, or by thermal dehydration of amino acid derivatives andhigh boiling solvents as described by Kopple et al., J. Org. Chem., 33(2), 862-864 (1968).

[0041] A typical synthesis of a diketopiperazine is illustrated in FIG.1 The COOH group(s) of an amino acid benzyl ester are activated in step1 to yield a protected ester. The amine is deprotected and cyclized viadimerization in step, 2, providing a diketopiperazine di-ester. Finallyin step 3, the COOH group(s) are deprotected to provide thediketopiperazine.

[0042] Ester derivatives of these diketopiperazine carriers are alsouseful in the present invention.

[0043] Delivery Systems

[0044] The carriers of the present invention are pharmacologicallyharmless, as are the microspheres prepared therefrom. They do noteffectively impair the active (i.e. biological, chemical, therapeutical,pharmacological, or the like) agent.

[0045] The diketopiperazine carriers of the present invention may beused to prepare compositions for delivering active agent cargoes, andparticularly biologically active agent cargoes. Delivery compositionswhich include the active agent and the carrier may be in the form ofmixtures of active agent and carrier or the carrier may form amicrosphere which contains the active agent. The carrier describedherein facilitates the delivery of the cargo to a target.

[0046] Microspheres containing an active agent can generally be of thematrix form or the microcapsule form. The matrix form includes both ahollow matrix sphere in which the carrier forms a matrix shell around ahollow center and the active agent is distributed throughout the matrixand a solid matrix sphere in which the carrier forms a spherical matrixcontinuum in which the active agent is distributed.

[0047] The microcapsule form is one in which the encapsulated activeagent either is in solution or is a solid, with the carrier forming ashell around the encapsulated material. The microcapsule form is theform most often taken by the self assembly of the carriers of thepresent invention.

[0048] Delivery compositions may be mixtures which may be formulatedsimply by mixing the carrier with the active agent prior toadministration. If the delivery composition is to be of the microsphereform, carrier microspheres can be prepared by dissolving the carrier inan appropriate solute and then stimulating self assembly by contactingthe carrier solution with a precipitator. Solubility of the carrier canbe regulated by the selection of the appropriate diketopiperazine.Furthermore, the diketopiperazines, and therefore, the compositions ofthe present invention can be pH adapted to be selectively soluble inspecific acidic, basic, or neutral pH ranges.

[0049] Delivery compositions which are targeted to an acidic environmentcan be made selectively soluble at acidic pH, such as the pH in thestomach. These compositions are prepared with an acid-soluble carrier.The acid-soluble carrier exists largely in the cation form in at least aportion of the pH range from about 1 to about 6.8. However, above about6.8 or at selected ranges above pH 6.8, the carrier is largelyunprotonated and insoluble in water. Therefore, the carrier could selfassemble to microspheres at basic or neutral pH, and the active agent inthe delivery composition would not be released until the carriersolubilizes upon encountering an acidic pH.

[0050] Delivery compositions which are to be targeted to an alkalineenvironment can be made selectively soluble at alkaline pH, such as thepH in the distal portion of the intestine. These compositions areprepared with a base-soluble carrier. The base-soluble carrier existslargely in an anionic form in at least a portion of the pH range of fromabout 7.2 to about 11. However, below and at pH 7.2, the carrier islargely protonated and insoluble in water. Therefore, the carrier couldself assemble to microspheres at acidic or neutral pH, and the activeagent in the delivery composition would not be released until thecarrier solubilizes upon encountering a basic pH.

[0051] Delivery compositions which are targeted to a neutral environmentcan be made selectively soluble at neutral pH. These compositions areprepared with a neutral-soluble carrier. The neutral-soluble carrierexists largely in a neutral form at neutral pH, i,e. from about 6.8 toabout 7.2. However, above or below this range, the carrier is insolublein water. Therefore, the carrier could self assemble to microspheres atacidic or basic pH, and the active agent in the delivery compositionwould not be released until the carrier solubilizes upon encountering aneutral pH.

[0052] In a typical formulation, the final solution can contain fromabout 10 mg to about 2000 mg of carrier per nil of solution, preferablybetween about 75 to about 500 mg of carrier per nil of solution, andmost preferably from about 75 to about 200 mg per nil. Optionally, themixture is heated to a temperature between about 20° C. and about 60°C., preferably about 40° C., until the carrier dissolves. Particulatesremaining in the solution may be filtered out by conventional means suchas gravity filtration over filter paper. The carrier solution usually ismaintained at the elevated temperature and is mixed with the activeagent and a precipitator, for example, an acid solution such as, forexample, aqueous acetic or citric acid at a concentration ranging fromabout 1N to about 3N for acid insoluble carriers, a basic solution forbase insoluble carriers, and a neutralizing solution for neutralinsoluble carriers. The active agent can be mixed with the precipitatingsolution or can be used separately. The resultant mixture is maintainedfor a period of time sufficient for microsphere formation as observed bylight microscopy. Although it is preferred that the precipitatingsolution is added to the carrier solution, the carrier solution can beadded to the precipitating solution as well.

[0053] The solutions above may optionally contain additives such asstabilizing additives. The presence of such additives promotes thestability and dispersability of the active agent in solution. Thestabilizing additives may be employed at a concentration ranging betweenabout 0.1 and 5% (w/v), preferably about 0.5% (w/v). Suitable, butnon-limiting examples of stabilizing additives include buffer salts, gumacacia, gelatin, methyl cellulose, polyethylene glycol, and polylysine.The preferred stabilizing agents are gum acacia, gelatin, and methylcellulose.

[0054] The amount of active agent which may be encapsulated by themicrosphere is dependent upon a number of factors which include theconcentration of agent in the encapsulating solution as well as theaffinity of the agent for the carrier. The concentration of the activeagent in the final formulation also will vary depending on the requireddosage of treatment. When necessary, the exact concentration can bedetermined by, for example, reverse phase HPLC analysis. When thepresent compositions are in microsphere form, the particle size of themicrosphere can also aid in providing efficient delivery of the activeagent to the target. Typically, microspheres of the present inventionwill have a diameter of less than 10 μm, preferably in the range of fromabout 0.1 μm to about 10 μm, and most preferably in the range of from0.2 μm to about 10 μm. The size of the microspheres containing an activeagent can be controlled by manipulating a variety of physical orchemical parameters, such as the pH, osmolarity, ionic strength of theencapsulating solution, or size of the ions in solution, and/or by thechoice of the precipitator used in the microsphere forming and loadingprocess.

[0055] For example, in the GI tract it is often desirable to usemicrospheres which are sufficiently small to deliver effectively theactive agent at the targeted area within the gastrointestinal tract.Small microspheres can also be administered parenterally by suspendingthe spheres in an appropriate carrier fluid (e.g. isotonic solution) andinjecting the solution directly into the circulatory system,intramuscularly, or subcutaneously. The mode of administration of thedelivery compositions will vary, of course, depending upon therequirement of the active agent administered. It has been noted thatlarge amino acid microspheres (greater than 50 μm) tend to be lesseffective as oral delivery systems.

[0056] The delivery compositions of the present invention may alsoinclude one or more enzyme inhibitors. Such enzyme inhibitors include,but are not limited to, compounds such as actinonin or epiactinonin andderivatives thereof. These compounds have the formulas below:

[0057] Derivatives of these compounds are disclosed in U.S. Pat. No.5,206,384. Actinonin derivatives have the formula:

[0058] wherein R¹² is sulfoxymethyl or carboxyl or a substituted carboxygroup selected from carboxamide, hydroxyaminocarbonyl and alkoxycarbonylgroups; and R¹³ is hydroxyl, alkoxy, hydroxyamino or sulfoxyamino group.Other enzyme inhibitors include, but are not limited to, aprotinin(Trasylol) and Bowman-Birk inhibitor.

[0059] The delivery compositions of the present invention may beformulated into dosage units by the addition of one or moreexcipient(s), diluent(s), disintegrant(s), lubricant(s), plasticizer(s),colorant(s), or dosing vehicle(s). Preferred dosage unit forms are oraldosage unit forms. Most preferred dosage unit forms include, but notlimited to, tablets, capsules, or liquids. The dosage unit forms caninclude biologically, pharmacologically, or therapeutically effectiveamounts of the active agent or can include less than such an amount ifmultiple dosage unit forms are to be used to administer a total dosageof the active agent. Dosage unit forms are prepared by methodsconventional in the art.

[0060] The compositions of the subject invention are useful foradministering biologically active agents to any animals such as birds;mammals, such as primates and particularly humans; and insects. Thesystem is particularly advantageous for delivering chemical orbiologically active agents which would otherwise be destroyed orrendered less effective by conditions encountered before the microspherereaches its target zone (i.e. the area in which the active agent of thedelivery composition are to be released) and within the body of theanimal to which they are administered. Particularly, the compositions ofthe present invention are useful in orally administering active agents,especially those which are not ordinarily orally deliverable.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0061] The following examples illustrate the invention withoutlimitation.

[0062] Reagents were purchased from Sigma Chemical Co.—St. Louis, Mo.,and were used without further purification. Flash column chromatographywas performed on Silica gel 40 mm, obtained from J. T. Baker—Co. NMRspectra were recorded on Varian EM-390, VXR-300, or QE-300 instrumentsand were run with chemical shifts given in pats per million downfieldfrom an internal tetramethylsilane or sodium3-(trimethylsilyl)-propionate standard. Mass spectra were obtained on aKratos MS 80RFA or a Finnigan 4516 MS instrument. All optical rotationswere performed at 589 nm (the Na D-line) at 22° C. on a Perkin-Elmer 241polarimeter, with “c” expressed as g of compound per 100 ml of solvent.Melting points are uncorrected.

EXAMPLE 1 Diketopiperazine of L-glutamic Acid

[0063] NαBOC-γ-benzyl-L-glutamic acid (NBGA) (6.0 g, 17.8 mmol) andN-hydroxy succinimide (2.25 g, 19.6 mmol) were dissolved in anhydroustetrahydrofuran (THF) (150 mL). The solution was cooled to 0° C. in anice bath and dicyclollexylcarbodimide (DCC) (4.04 g, 19.6 mmol)dissolved in 40 mL anhydrous THF was added dropwise over 30 minutes. Theice bath was removed. The solution was allowed to warm to roomtemperature and was stirred overnight. The reaction was monitored bythin layer chromatography (TLC) (20% EtOH/CHCl₃).

[0064] When the reaction was completed, the solution was filtered andthe filtrate was concentrated to provide crude N-hydroxy succinimide(NHS) ester of NαBOC-γ benzyl-L-glutamic acid as a viscous semi-solid(8.7 g).

[0065] Trifluoroacetic acid (TFA, 1.3 mL) was added dropwise to aportion of this NHS ester (0.50 g, 1.02 mmol) at 0° C. The solution wasslowly allowed to warm to room temperature and was stirred overnight.

[0066] The volatile material was removed at reduced pressure, and acrude yellow solid (0.85 g) was recrystallized from EtOAc to providepure diketopiperazine L-glutamic acid dibenzyl ester (0.11 g, 50%).

[0067] Properties of the diketopiperazine are listed below. m.p.275-277° C. ¹H NMR (d₆-DMSO): δ 8.26 (s,2H,NH), 7.46 (s, 10H, aromatic),5.16 (s, 4H, CH₂), 3.98 (t, 2H, CH), 2.58 (m, 4H, CH2), 2.06 (m, 4H,CH2). Analysis: Calc. for C₂₄H₂₆N₂O₆: C 66.74, H 5.98, N 6.39:  Found: C65.73, H 6.03, N 6.35. Mass spectrum: Theoretical: 438.18;  Found: 439(M + 1). Optical rotation: [α]_(D) −23.4° (c = 1, dioxane).

EXAMPLE 2 Diketopiperazine of L-glutamic Acid

[0068] The diketopiperazine of L-glutamic acid dibenzyl ester wasprepared according to the method of Example 1 (0.90 g, 2.05 mmol, 4.1mequiv.) and was dissolved in a mixture of EtOAc/MeOH (6:1, 470 ml).Pd-C (0.20 g) catalyst was added The black suspension was degassed threetimes, and hydrogen gas was introduced. The reaction was monitored byTLC (30% EtOH/CHCl₃).

[0069] The catalyst was filtered off, and the resultant diacidprecipitate was washed five times with boiling MeOH and EtOAc todissolve the diacid. The filtrate was concentrated to provide thediketopiperazine of L-glutamic acid as a white solid (0.53 g, 100%).

[0070] Properties of the diketopiperazine are listed below: m.p.234-236° C. ¹H NMR (d₇-DMF): δ 4.00 (t, 2H, CH), 2.49 (m, 4H, CH₂), 2.10(m, 4H CH₂). Analysis: Calc. for: C₁₀H₁₄N₂O₆: C 46.51; H 5.46; N 10.85:  Found: C 46.72; H 5.50; N 10.82. High resolution mass spectrum:Theoretical: 259.0930 (M + H);  Found: 259.033 (M + H). Opticalrotation: [α]_(D) −52° (c = 1, DMSO).

EXAMPLE 3 Diketopiperazine of L-aspartic Acid

[0071] The method of Example 1 was followed, substitutingβ-benzyl-Nα-BOC-L-aspartic acid (24.0 g, 74.2 mmol) for the NBGA, 9.40 g(81.7 mmol) of the NHS, and 16.85 g (81.7 mmol) of the DDC in anhydrousTHF to provide 37.13 g of crude NHS ester.

[0072] This NHS ester (37.13 g) was reacted with TFA (85 ml) at 0° C. toyield a crude TFA salt. The salt was neutralized in drydimethylformamide (DMF) (100 mL) and pyridine (3.5 L) at 0° C.Recrystallization from EtOAc provided the diketopiperazine of L-asparticacid dibenzyl ester as a white solid (7.13 g, 47%) m.p. 157° C.

[0073] Properties of the diketopiperazine are listed below. ¹H NMR(CDCl₃) δ 7.31 (s, 10H, aromatic) 6.72 (s, 2H, NH), 5.12 (s, 4H, CH₂),4.35 (m, 2H, CH), 3.00 (m, 4H, CH₂). Analysis: Calc. for C₂₂H₂₂N₂O₆: C64.38; H 5.40; N 6.83:  Found: C 64.27; H 5.39; N 6.79. High resolutionmass spectrum: Theoretical: 410.1478: Found: 410.1503. Optical rotation:[α]_(D) −69.50′ (c = 1, CHCL₃).

EXAMPLE 4 Diketopiperazine of L-aspartic Acid

[0074] The diketopiperazine of L-aspartic acid dibenzyl ester (6.15 g,15 mmol, 30 mequiv.) was prepared according to the method of Example 3and was dissolved in MeOH (250 mL). Pd-C (0.90 g) catalyst was added.The black suspension was degassed three times, and hydrogen gasintroduced. The reaction was monitored by TLC (30% EtOH/CHCl₂).

[0075] The catalyst was filtered off, and resultant diacid precipitatewas washed five times with boiling MeOH to dissolve the diacid. Thefiltrate was concentrated to provide a white solid which was rinsed withMeOH and dried to provide the diketopiperazine of L-aspartic acid as awhite solid (2.78 g, 80%).

[0076] Properties of the diketopiperazine are listed below. m.p.254-255° C. ¹H NMR (CDCl₃-d₆ DMSO, 1:1 by vol) δ 7.80 (s, 2H, NH), 4.20(t, 2H, CH), 2.82 (D, 4H, CH₂). Analysis: Calc. for C₈H₁₀N₂O₆: C 41.75;H 4.38; N 12.17:  Found: C 41.82; H 4.39; N 12.09. Optical rotation:[α]_(D) −37° (c = 1, DMSO).

EXAMPLES 5-8

[0077] The diketopiperazines prepared according to the methods ofExamples 1-4 (0.1 mmol) are dissolved in 0.1 ml of aqueous Li₂CO₃ (1M)deionized water to provide a clear solution of the lithium salt. 50 μlof this 1 M solution are mixed with 50 μl of 0.86M citric acid. Themixture is shaken to yield a white suspension. Microspheric examinationof the suspension reveals the presence of tiny spheres which moverandomly throughout the field of inspection. Spheres ranging in size upto about 10 μl are observed.

EXAMPLES 9-12 Preparation of Diketopiperazine Microspheres ContainingEncapsulated Salmon Calcitonin

[0078] Diketopiperazines prepared according to the methods of Examples1-4 are dissolved at 40° C. in distilled water (640 μL) with 100 μl ofTris base tris(hydroxymethylamine) in distilled water, to prepare asolution having a carrier concentration of 50 mg/ml. Water is added tobring the total volume to 4.0 ml. The sample has a carrier concentrationof 200 mg/mL. Salmon calcitonin (6 μg) and 2M citric acid are added tothe solution. The total salmon calcitonin concentration is 1.5 μg/mL.Microspheres containing salmon calcitonin are observed.

EXAMPLES 13-16 In Vivo Evaluation of Calcitonin Preparations in Rats

[0079] Six fasted rats are anesthetized. The rats are administered, byoral gavage, diketopiperazine/calcitonin compositions containing 1.5 μgof calcitonin/ml prepared by the methods of Examples 9-12. Each rat isadministered a dosage of 10 μg/kg. The amount of diketopiperazine in thedosage is 300 mg/kg.

[0080] Blood samples are collected serially from the tail artery. Serumcalcium is determined by testing with a Demand™ Calcium Kit (SigmaChemical Company—St. Louis, Mo.).

COMPARATIVE EXAMPLE 13A In Vivo Evaluation of Calcitonin Preparations inRats

[0081] A second group of rats is administered, by oral gavage, 10 μg/kgof salmon calcitonin without any carrier.

EXAMPLES 17-20 In Vivo Evaluation of Interferon Preparations in Rats

[0082] A dosing preparation is prepared containing interferon α2b anddiketopiperazine carriers prepared according to the methods of Examples1-4 in a Trizma® hydrochloride buffer solution (Tris-HCl) at a pH ofabout 7-8.

[0083] The samples containing the interferon α2b and carrier areadministered by oral gavage, to five rats. The dose is 1000 μg/kg. Theamount of carrier is 800 mg/kg. Delivery is evaluated by using an ELISAassay (BioScience Int.'l.—Camarillo, Calif.) for human interferon a.

EXAMPLES 21-24 Toxicity Studies

[0084] Male mice (BALB/c) are fed a dose of 1 g/kg of thediketopiperazine prepared according to the methods of Examples 1-4 perday for five (5) days. The compound shows neither acute nor chronictoxicity (over the 5 day period), and no unusual behavior is observed.

EXAMPLE 25-28 In vitro Enzyme Kinetics of Pancreatin Digestion of SalmonCalcitonin

[0085] The following solutions are prepared:

[0086] Salmon calcitonin (sCt), 10 mg/nl in 0.085 N citric acid;potassium phosphate (monobasic), 7 mg/ml (titrated to pH 7 with 1 NNaOH); pancreatin, 20 mg/ml in potassium phosphate solution;diketopiperazine carriers are prepared by dissolving carriers preparedaccording to the methods of Examples 1-4 in potassium phosphatesolution, titrating to pH 7.2±0.1 (1 N NaOH or HCl as needed), heatingto 37° C., stirring, and filtering through 0.2μ syringe filter.

[0087] Eight 1.7 ml eppendorf tubes are prepared. Two ml of the carriersolution are placed in several 5 ml stoppered tubes. Two ml of potassiumphosphate solution are placed in control tubes (5 ml). 100 μl of sCTstock solution are added to each 5 ml tube. The solutions are vortexed,and a 100 μl aliquot of each tube is transferred to the first eppendorftube in each set (baseline). The eppendorf tubes are immediately cooledto −78° C. in a dry ice/acetone bath and stored for analysis at a latertime. 100 μl of pancreatin stock solution are added to each tube. Thetubes are vortexed. 100 μl of the solution are transferred to a secondeppendorf tube and are frozen. The 5 ml tubes with the reagents areplaced in a 37° C. water bath for one hour. Samples are obtained at thefollowing times 0 min. (baseline), 0.1 min., 1 min., 5 min., 10 min., 15min., 30 min., and 60 min. Samples are kept at −70° C. until ready forassay.

[0088] The samples are assayed using HPLC to determine the amount ofcalcitonin remaining. The conditions are as follows: Column: RANIN C4 3cm × 4.6 mm, 10 μm particle size, 300 Å pore size (Solvent) Mobile PhaseA: 10% CH₃CN/90% H₂O in 20 mM potassium phosphate buffer at pH 7 MobilePhase B: 60% CH₃CN/40% H₂O in 20 mM potassium phosphate buffer at pH 7Pump: Hitachi L-6200 Intelligent Pump Linear Gradient: STEP TIME (min) AB 1 0 70%  30% 2 7 40%  60% 3 7.1 — 100% 4 80 — 100% 5 8.1 70%  30% FlowRate: 2.5 ml/min

[0089] Step 1-2 is a linear gradient from 70%A/30%B to 40%A/60%B. Steps2-3 is a direct charge to 100%B for 0.9 min followed by a direct chargeto 70%A/30%B at 8.1 min. Detector: UV 220 nm

COMPARATIVE EXAMPLE 25A

[0090] The procedure of Examples 25-28 is followed omitting the additionof carrier solution to the eppendorf tubes.

[0091] All patents, applications, test methods, and publicationsmentioned herein are hereby incorporated by references.

[0092] Many variations of the present invention will suggest themselvesto those skilled in the art in light of the above detailed disclosure.All such modifications are within the full intended scope of theappended claims.

In the claims:
 1. A delivery composition comprising: (a) an activeagent; and (b) at least one diketopiperazine selected from the groupconsisting of: (i) a mono C-substituted diketopiperazine and (ii) adi-C-substituted diketopiperazine.
 2. A delivery composition as definedin claim 1, comprising a microsphere.
 3. A delivery composition asdefined in claim 2, wherein said microsphere comprises a microcapsule.4. A delivery composition as defined in claim 2, wherein saidmicrosphere has a diameter of less than about 10 μm.
 5. A deliverycomposition as defined in claim 1, wherein said active agent comprises afragrance.
 6. A delivery composition as defined in claim 1, wherein saidactive agent comprises a biologically active agent.
 7. A deliverycomposition as defined in claim 6, wherein said biologically activeagent is selected from the group consisting of a peptide, amucopolysaccharide, a carbohydrate, a lipid, a pesticide, or anycombination thereof.
 8. The delivery composition as defined in claim 7,wherein said biologically-active agent is selected from the groupconsisting of human growth hormone, bovine growth hormone, growthhormone-releasing hormone, an interferon, interleukin-II, insulin,heparin, calcitonin, erythropoietin, atrial naturetic factor, anantigen, a monoclonal antibody, somatostatin, adrenocorticotropin,gonadotropin releasing hormone, oxytocin, vasopressin, cromolyn sodium,vancomycin, desferrioxamine (DFO), or any combination of any of theforegoing.
 9. A delivery composition as defined in claim 8, wherein saidbiologically-active agent is selected from the group consisting of aninterferon, interleukin-II, insulin, heparin, calcitonin, oxytocin,vasopressin, cromolyn sodium, vancomycin, DFO, or any combination of anyof the foregoing.
 10. A delivery composition as defined in claim 1,wherein said diketopiperazine is derived from two α-amino acids.
 11. Adelivery composition as defined in claim 10, wherein said two a-aminoacids from which said diketopiperazine is derived are independentlyselected from the group consisting of glutamic acid, aspartic acid,tyrosine, phenylalanine, and optical isomers thereof.
 12. A deliverycomposition as defined in claim 10, wherein said two α-amino acids fromwhich said diketopiperazine is derived are the same.
 13. A deliverycomposition as defined in claim 10, wherein said diketopiperazine isprepared by the thermal condensation of said two α-amino acids fromwhich said diketopiperazine is derived.
 14. A delivery composition asdefined in claim 1, further comprising (c) at least one enzymeinhibitor.
 15. A pharmacological composition comprising: (a) at leastone pharmacologically active agent; and (b) at least onediketopiperazine selected from the group consisting of: (i) amono-C-substituted diketopiperazine and (ii) a di C-substituteddiketopiperazine.
 16. A dosage unit form comprising: (A) a deliverycomposition as defined in claim 1; and (B) (a) an excipient, (b) adiluent, (c) a disintegrant, (d) a lubricant, (e) a plasticizer, (f) acolorant, (g) a dosing vehicle, or (h) any combination thereof.
 17. Adosage unit form as defined in claim 15, comprising an oral dosage unitform.
 18. A method for administering a biologically active agent to ananimal in need of such agent, said method comprising administeringorally to said animal, a composition as defined in claim
 1. 19. A methodfor preparing microspheres containing an active agent, said methodcomprising: (A) solubilizing, in a solvent, at least onediketopiperazine selected from the group consisting of: (i) amono-C-substituted diketopiperazine and (ii) a di-N-substituteddiketopiperazine. to yield a diketopiperazine solution; and (B)contacting said diketopiperazine solution with said active agent and aprecipitator solution in which said diketopiperazine is insoluble.
 20. Amethod as defined in claim 19, wherein said diketopiperazine solutionhas a pH within a first range and said precipitator solution has a pHwithin a second range, said first range being different than said secondrange.